Cell morphology, migration, growth and differentiation result from both adhesion dependent and growth factor receptor mediated signaling events. Adhesion dependent signaling events link the extracellular matrix with the intracellular cytoskeleton both structurally and biochemically through the formation of macromolecular complexes at sites of cell matrix interactions. In cell culture, these macromolecular complexes are referred to as focal adhesions. Burridge and Chrzanowska-Wodnicka (1996) Ann. Rev. Cell. Dev. Biol. 12:463-518. The ability of adhesion receptors to mediate both signaling and structural events occurs, in part, through the association of their cytoplasmic domains with cytoskeletal components. The cytoskeletal components in turn provide structural and adapter functions for the assembly of the intracellular signaling complexes. Cytoskeletal proteins that provide structural and/or adapter functions include actin, alpha-actinin, paxillin, talin, tensin and vinculin. Associated signaling proteins include tyrosine kinases (focal adhesion kinase, src, csk and fyn), the serine-threonine kinase families of PKC and MAPK and members of the RAS family of small GTP binding proteins. Clark and Brugge (1996) Science 268:233-239; Burridge and Chrzanowska-Wodnicka (1996) Ann. Rev. Cell. Dev. Biol. 12:463-518.
Members of the syndecan family of cell surface heparan sulfate proteoglycans (syndecan-1 through 4) have been implicated in mediating cell adhesion and morphology. Bernfield et al. (1991) Ann. Rev. Cell Biol. 8:365-393; Carey (1997) Biochem. J. 327:1-16; Liu et al. (1998) J. Biol. Chem. 273:22825-22832; Woods and Couchman (1998) Trends Cell Biol. 8:189-192. All four syndecan family members contain a high degree of sequence conservation of their cytoplasmic domains, which, based on homology can be divided into conserved membrane proximal, variable central, and conserved C-terminal subdomains. Several cellular components which associate, either directly or indirectly, with the cytoplasmic domain of syndecan family members have been identified. These include: PKCxcex1 and phosphatidyl inositol 4,5-biphosphate (PIP2) which interact directly with the variable central region of the cytoplasmic domain of syndecan-4 (Oh et al. (1997) J. Biol. Chem. 272:11805-11811; Oh et al. (1997) J. Biol. Chem. 272:8133-8136; Oh et al. (1998) J. Biol. Chem. 273:10624-10629); the PDZ containing proteins syntenin (Grootjans et al. (1997) Prot. Natl. Acad. Sci. USA 94:13683-13688) and CASK/LIN-2 (Cohen et al. (1998) J. Cell Bio. 142:129-138; Hsueh et al. (1998) J. Biol. Chem. 142:139-151) which interact with the cytoplasmic domains of all syndecan family members through the highly conserved C-terminal EFYA sequence, and a Src-cortactin complex which associates with the membrane proximal domain of syndecan-3. Kinnunen et al. (1998) J. Biol. Chem. 273:10702-10708.
Syndecan-4 is observed in focal contacts (Woods and Couchmana (1994) Mol. Biol. Cell 5:183-192; Baciu and Goetinck (1995) Mol. Biol. Cell 1:1503-1513) and associates with PKCxcex1 (Oh et al. (1997) J. Biol. Chem. 272:11805-11811; Oh et al. (1997) J. Biol. Chem. 272:8133-8136).
The present invention is based, in part, on the discovery of a gene which encodes a novel cellular protein referred to herein as syndecan-4 binding protein (S4BP). The S4BP protein was found to interact with the cytoplasmic domain of syndecan-4 but not other members of the syndecan family. When S4BP is overexpressed, it mediates cell spreading and actin cytoskeleton organization. S4BP plays a role in linking syndecan-4 to the focal adhesion complex.
Accordingly, in one aspect, the invention features an isolated nucleic acid molecule (e.g., cDNAs) comprising a nucleotide sequence encoding an S4BP protein or a biologically active portion thereof, as well as, nucleic acid fragments suitable as primers or hybridization probes for the detection of S4BP-encoding nucleic acid (e.g., mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 1, or the coding region (SEQ ID NO:3), or a complement of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention includes a nucleotide sequence which hybridizes, preferably under stringent conditions, to or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO:1, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2. The preferred S4BP nucleic acid encodes a protein which also preferably possesses at least one of the S4BP activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2, e.g., sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains an S4BP biological activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to play a role in cell matrix interactions. In one embodiment, the protein encoded by the nucleic acid molecule has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86, 88, 90%, and most preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 (e.g., the entire amino acid sequence of SEQ ID NO:2). In another preferred embodiment, the protein is a full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by the open reading frame shown in SEQ ID NO:3). In another embodiment, the protein is a mammalian protein, e.g., a human protein, which is substantially homologous to the amino acid sequence of SEQ ID NO:2, or a portion thereof.
In yet another embodiment, the isolated nucleic acid molecule encodes a portion of an S4BP protein which includes a sequence encoding a SH3 domain binding site motif. Preferably, the SH3 domain binding site motif encoded by the nucleic acid molecule has at least about 80% or more sequence identity to the SH3 domain binding site motif (i.e., about amino acid residues 24 to 27) of SEQ ID NO:2. Preferably, the SH3 domain binding site motif has a consensus sequence Pro-Xaa-Pro-Pro, where Xaa is any amino acid.
In another preferred embodiment, the isolated nucleic acid molecule encodes an S4BP protein or portion thereof which has at least about 55% or more sequence identity to SEQ ID NO:2 and has one or more of the following activities involved with cell matrix interactions: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with paxillin; 3) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with PKCxcex1; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; and/or 9) it modulates cell spreading.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes naturally-occurring S4BP or a biologically active portion thereof. Preferably, the biologically active portion is preferably encoded by a nucleotide sequence greater than 150, 200, 300, 400, 500, 600, 700 or 1000 base pairs in length. Moreover, given the disclosure herein of S4BP-encoding cDNA sequences (e.g., SEQ ID NO:1), antisense nucleic acid molecules (i.e., molecules which are complementary to the coding strand of the S4BP cDNA sequence) are also provided by the invention.
In a preferred embodiment, the encoded S4BP protein differs in amino acid sequence at least 1 to as many as (but not more than) 2, 3, 5, 10, 20 or 40 residues from a sequence in SEQ ID NO:2. In a preferred embodiment, the differences, however, are such that: the S4BP encoded protein exhibits an S4BP biological activity, e.g., the encoded S4BP protein retains a biological activity of a naturally occurring S4BP, e.g., the S4BP protein of SEQ ID NO:2.
In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2, fused, e.g., in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:2.
In preferred embodiments the encoded S4BP protein includes an S4BP sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In another aspect, the invention features vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce S4BP protein by culturing the host cell in a suitable medium. The S4BP protein can be then isolated from the medium or the host cell.
In yet another aspect, the invention features a transgenic nonhuman animal, e.g., a rodent (e.g., a mouse), or a cow, goat, pig, rabbit or guinea pig, in which an S4BP gene has been introduced or altered. In one embodiment, the genome of the nonhuman animal has been altered by introduction of a nucleic acid molecule of the invention encoding S4BP as a transgene. In another embodiment, an endogenous S4BP gene within the genome of the nonhuman animal has been altered, e.g., functionally disrupted, by homologous recombination.
In still another aspect, the invention features an isolated S4BP protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated S4BP protein or portion thereof plays a role in cell matrix interaction. In another preferred embodiment, the isolated S4BP protein or portion thereof is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains one or more S4BP activity.
In one embodiment, the biologically active portion of the S4BP protein includes a domain or motif, preferably a domain or motif which has an S4BP activity. The motif can be, e.g., a SH3 binding domain site motif, and/or at least one or two myristoylation motif(s).
The invention also provides an isolated preparation of an S4BP protein. In preferred embodiments, the S4BP protein includes the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by the open reading frame shown in SEQ ID NO:3). In yet another embodiment, the protein has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86, 88, 90%, and most preferably at least about 90-95% 96%, 97%, 98% or 99% sequence identity to the entire amino acid sequence of SEQ ID NO:2. In other embodiments, the isolated S4BP protein includes an amino acid sequence which has at least about 60-70% or more sequence identity to the amino acid sequence of SEQ ID NO:2 and has an one or more of the following activities: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with paxillin; 3) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with PKCAxcex1; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
Alternatively, the isolated S4BP protein can include an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95% 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence of SEQ ID NO:1. It is also preferred that the preferred forms of S4BP also have one or more of the S4BP activities described herein.
In a preferred embodiment, the S4BP protein differs in amino acid sequence at least up to 1 to as many (but not more than) 2, 3, 5, 10, 20, or 40 residues, from a sequence in SEQ ID NO:2. In other preferred embodiments, the S4BP protein differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:2. Preferably, the differences are such that: the S4BP protein exhibits an S4BP biological activity, e.g., the S4BP protein retains a biological activity of a naturally occurring S4BP.
In another aspect, the invention features a fragment of an S4BP protein capable of binding to syndecan-4. In a preferred embodiment, the fragment comprises at least 10, 15, 20, 25, 30, 50, 100, 150 amino acid residues and is capable of binding to syndecan-4.
In another aspect of the invention, the S4BP protein is a recombinant S4BP protein which differs from S4BP isolated from tissue in one or more of the following: its pattern of glycosylation, myristoylation, phosphorylation, or other posttranslational modifications.
Another aspect of the invention features a fragment of a syndecan-4 protein which is capable of binding to syndesmos. In a preferred embodiment, the fragment comprises at least 10, 15, 20, 25, 30 amino acid residues and is capable of binding to syndesmos. Preferably, the syndesmos binding fragment comprises at least 10, 15, 17, 20 or 30 but not more than 20, 28, 30, 40, 50, or 100 amino acid residues of syndecan-4. In a preferred embodiment, the syndesmos binding fragment comprises amino acids 169 to 197 of SEQ ID NO:2 (SEQ ID NO:6) or 180 to 197 of SEQ ID NO:2.
The S4BP protein of the invention, or portions or fragments thereof, can be used to prepare anti-S4BP antibodies. Accordingly, the invention also provides an antigenic peptide of S4BP which includes at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of S4BP such that an antibody raised against the peptide forms a specific immune complex with S4BP. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30, 50, 70, 80 amino acid residues. The invention further provides an antibody, e.g., a monoclonal antibody that specifically binds S4BP. In one embodiment, the antibody is monoclonal. In another embodiment, the antibody is coupled to a detectable substance. In yet another embodiment, the antibody is incorporated into a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.
In another aspect, the S4BP binding fragment of syndecan-4, can be used to prepare antibodies. Accordingly, the invention also provides an antigenic peptide of syndecan-4 which includes at least 8 to 17 amino acid residues of the amino acid sequence shown in SEQ ID NO:5 and encompasses an epitope of syndecan-4 such that an antibody raised against the peptide forms a specific immune complex with S4BP binding region of syndecan-4. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30, 50 amino acid residues. The invention further provides an antibody, e.g., a monoclonal antibody that specifically binds S4BP binding region of syndecan-4. In one embodiment, the antibody is monoclonal. In another embodiment, the antibody is coupled to a detectable substance. In yet another embodiment, the antibody is incorporated into a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.
In another aspect, the invention features, a method of modulating an S4BP mediated property of a cell, in vitro or in vivo. The method includes contacting the cell with an agent which modulates, e.g., inhibits the interaction of S4BP with syndecan-4.
The S4BP/syndecan-4 interaction can be inhibited or reduced by administering an agent which interferes with the binding of S4BP and syndecan-4. Examples of such agents include an antibody, e.g., an intrabody, e.g., an antibody, which binds to S4BP or an antibody which binds to syndecan-4 (e.g., an antibody which binds the S4BP binding region of syndecan-4), or both. Other agents include: an S4BP protein, or a syndecan-4 binding portion thereof; a syndecan-4 protein, or an S4BP binding portion thereof; a fusion of an S4BP protein, or a syndecan-4 binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a fusion of a syndecan-4 protein, or an S4BP binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a polypeptide other than S4BP or syndecan-4 which binds to S4BP or syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method: modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); modulates actin stress fiber formation and/or organization; modulates adhesion formation; modulates cell attachment; modulates cell spreading.
In a preferred embodiment, the method includes treating a subject having a disorder characterized by unwanted or aberrant S4BP protein activity or nucleic acid expression.
In a preferred embodiment, the method includes treating a subject having a disorder associated with unwanted or abnormal cellular interactions (e.g., unwanted or abnormal cell-cell and/or cell-matrix interactions, unwanted or abnormal cell migration/movement, e.g., cancer).
In a preferred embodiment, one or more of the following biological activities of S4BP is modulated: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with paxillin; 3) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with PKCxcex1; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
In another aspect, the invention features, a method of modulating an S4BP mediated property of a cell, in vitro or in vivo. The method includes contacting the cell with an agent which modulates the activity of S4BP. S4BP activity can be modulated, e.g., transcriptionally, translationally, or post-translationally.
In a preferred embodiment, S4BP activity is modulated by administering: an S4BP antisense molecule; an antibody, e.g., an intrabody, which binds to S4BP; syndecan-4 protein, or an S4BP binding portion thereof; paxillin, or an S4BP binding portion thereof; fusions of a syndecan-4 protein or paxillin, or an S4BP binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a polypeptide other than syndecan-4 or paxillin which bind to S4BP, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay, a small molecule, e.g., a small molecule which binds to the control region of S4BP. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method: modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); modulates actin stress fiber formation and/or organization; modulates adhesion formation; modulates cell attachment; modulates cell spreading.
In a preferred embodiment, the method includes treating a subject having a disorder characterized by unwanted or aberrant S4BP protein activity or nucleic acid expression.
In a preferred embodiment, the method includes treating a subject having a disorder associated with unwanted or abnormal cellular interactions (e.g., unwanted or abnormal cell-cell and/or cell-matrix interactions, unwanted or abnormal cell migration/movement, e.g., cancer).
In a preferred embodiment, one or more of the following biological activities of S4BP is modulated: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 3) it interacts, directly or indirectly, with PKCxcex1; 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with paxillin; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
In a preferred embodiment, the agent which modulates S4BP activity can be an agent which increases S4BP protein activity or S4BP nucleic acid expression. Examples of agents which increase S4BP protein activity or S4BP nucleic acid expression include small molecules (e.g., small molecules which bind to the promoter region of S4BP), active S4BP proteins, and nucleic acids encoding S4BP that have been introduced into the cell. In another embodiment, the agent which modulates S4BP activity can be an agent which decreases S4BP protein activity or S4BP nucleic acid expression. Examples of agents which inhibit S4BP activity or expression include small molecules, antisense S4BP nucleic acid molecules, and antibodies or intrabodies that specifically bind to S4BP or to its target syndecan-4 or paxillin. In a preferred embodiment, the cell is present within a subject and the agent is administered to the subject.
In another aspect, the invention features, a method of treating a subject having a disorder characterized by unwanted or abnormal cell adhesion or cell spreading (e.g., cancer). The method includes contacting the cell with an agent which modulates, e.g., inhibits, the interaction of S4BP with syndecan-4. In another embodiment, the method includes contacting the cell with an agent which modulates, e.g., inhibits, the interaction of S4BP with paxillin.
The S4BP/syndecan-4 interaction can be inhibited or reduced by administering an agent which interferes with the binding of S4BP and syndecan-4. Examples of such agents include an antibody, e.g., an intrabody, e.g., an antibody, which binds to S4BP or an antibody which binds to syndecan-4 (e.g., an antibody which binds the S4BP binding region of syndecan-4), or both. Other agents include: an S4BP protein, or a syndecan-4 binding portion thereof; syndecan-4 protein, or an S4BP binding portion thereof; a fusion of an S4BP protein, or a syndecan-4 binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a fusion of a syndecan-4 protein, or an S4BP binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a polypeptide other than S4BP or syndecan-4 which binds to S4BP or syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents
In a preferred embodiment, the method: modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); modulates actin stress fiber formation and/or organization; modulates adhesion formation; modulates cell attachment; modulates cell spreading.
In another aspect, the invention features, a method of treating a subject having a disorder characterized by unwanted or abnormal cell spreading (e.g., cancer). The method includes contacting the cell with an agent which modulates the activity of S4BP. S4BP activity can be modulated, e.g., transcriptionally, translationally, or post-translationally.
In a preferred embodiment, S4BP activity is modulated by administering: an S4BP antisense molecule; an antibody, e.g., an intrabody, which binds to S4BP; syndecan-4 protein, or an S4BP binding portion thereof; fusions of a syndecan-4 protein, or an S4BP binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes entrance into the cell or solubility; a polypeptide other than syndecan-4 which binds to S4BP, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay, a small molecule, e.g., a small molecule which binds to the control region of S4BP. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment the method: modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); modulates actin stress fiber formation; modulates adhesion formation; modulates cell attachment; modulates cell spreading.
In a preferred embodiment, one or more of the following biological activities of S4BP is modulated: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with paxillin; 3) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine kinase); 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with PKCxcex1; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
The agent which modulates S4BP activity can be an agent which increases S4BP protein activity or S4BP nucleic acid expression. Examples of agents which increase S4BP protein activity or S4BP nucleic acid expression include small molecules e.g., small molecules which bind to the promoter region of S4BP), active S4BP proteins, and nucleic acids encoding S4BP that have been introduced into the cell. In another embodiment, the agent which modulates S4BP activity can be an agent which decreases S4BP protein activity or S4BP nucleic acid expression. Examples of agents which inhibit S4BP activity or expression include small molecules, antisense S4BP nucleic acid molecules, and antibodies or intrabodies that specifically bind to S4BP or to its target syndecan-4. In a preferred embodiment, the cell is present within a subject and the agent is administered to the subject.
In a preferred embodiment, the method includes modulating cell attachment and/or cell spreading.
The invention also features methods for evaluating a subject at risk for a disorder. The method includes evaluating, e.g., detecting, a genetic lesion in the S4BP gene, or evaluating, e.g., detecting, misexpression of the S4BP gene, thereby determining if a subject is at risk for (e.g., has or is predisposed to have) a disorder. The disorder can be one which is characterized by aberrant or abnormal S4BP nucleic acid expression and/or S4BP protein activity, e.g., a disorder associated with abnormal cellular interactions (e.g., abnormal cell-cell and/or cell-matrix interactions, abnormal cell migration/movement). In a preferred embodiment, the method includes evaluating, e.g., in a sample of cells from the subject, the presence or absence of a genetic lesion, e.g., a lesion characterized by an alteration affecting the gene encoding an S4BP protein, or evaluating the misexpression of the S4BP gene. Genetic lesions can be evaluated, e.g., by contacting the sample with a nucleic acid probe capable of hybridizing to S4BP mRNA, e.g., a labeled probe. Expression can be evaluated with an antibody capable of binding to S4BP protein, e.g., a labeled antibody. In a preferred embodiment, the method can also be used in fetal or neonatal diagnosis.
In another aspect, the invention features evaluating, e.g., detecting, a genetic lesion in the syndecan-4 gene, thereby determining if a subject with the lesion is at risk for (e.g., has or is predisposed to have) a disorder characterized by unwanted or abnormal S4BP/syndecan-4 interaction. In one embodiment, the methods include evaluating, e.g., in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by an alteration affecting the gene encoding a syndencan-4 protein or evaluating the misexpression of the syndencan-4 gene. In a preferred embodiment, the methods include evaluating the presence or absence of a genetic lesion affecting the nucleotide sequence encoding the S4BP binding region of syndecan-4. Genetic lesions can be evaluated, e.g., by contacting the sample with a nucleic acid probe capable of hybridizing to syndecan-4 mRNA, e.g., a labeled probe. Expression can be evaluated with an antibody capable of binding to syndecan-4 protein, e.g., an antibody which binds the S4BP binding region of syndecan-4, e.g., a labeled antibody. In a preferred embodiment, the method can also be used in fetal or neonatal diagnosis
Another aspect of the invention features methods for detecting the presence of S4BP nucleic acid or protein in a biological sample. In a preferred embodiment, the method involves contacting a biological sample (e.g., a cell sample) with a compound or an agent capable of detecting S4BP protein or S4BP nucleic acid, e.g., mRNA, such that the presence of S4BP nucleic acid or protein is detected in the biological sample. The compound or agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to S4BP mRNA or a labeled or labelable antibody capable of binding to S4BP protein. The invention further provides methods for diagnosis of a subject with, for example, a disorder associated with abnormal cellular interactions (e.g., abnormal cell-cell and/or cell-matrix interactions, abnormal cell migration, movement, e.g., cancer) based on detection of S4BP protein or MRNA. In one embodiment, the method involves contacting a cell or tissue sample (e.g., a biopsy sample) from the subject with an agent capable of detecting S4BP protein or MRNA, determining the amount of S4BP protein or MRNA expressed in the cell or tissue sample, comparing the amount of S4BP protein or mRNA expressed in the cell or tissue sample to a control sample and forming a diagnosis based on the amount of S4BP protein or MRNA expressed in the cell or tissue sample as compared to the control sample. Specific diagnostic tests are described in greater detail below. Kits for detecting S4BP nucleic acid or protein in a biological sample are also within the scope of the invention and are described in greater detail below.
Still another aspect of the invention features methods, e.g., screening assays, for identifying a compound for treating a disorder characterized by aberrant S4BP nucleic acid expression and/or protein activity, e.g., a disorder associated with unwanted or abnormal cellular interactions (e.g., unwanted or abnormal cell-cell and/or cell-matrix interactions, unwanted or abnormal cell migration/movement). These methods typically include assaying the ability of the compound or agent to modulate the expression of the S4BP gene or the activity of the S4BP protein, thereby identifying a compound for treating a disorder characterized by aberrant S4BP nucleic acid expression and/or protein activity. In a preferred embodiment, the method involves contacting a biological sample, e.g., a cell or tissue sample, obtained from a subject having the disorder with the compound or agent, determining the amount of S4BP protein expressed and/or measuring the activity of the S4BP protein in the biological sample, comparing the amount of S4BP protein expressed in the biological sample and/or the measurable S4BP biological activity in the cell to that of a control sample. An alteration in the amount of S4BP protein expression and/or S4BP activity in the cell exposed to the compound or agent in comparison to the control is indicative of a modulation of S4BP expression and/or S4BP activity.
The invention also features methods for identifying a compound or agent which interacts with an S4BP protein. In a preferred embodiment, the interaction with an S4BP protein can be binding, phosphorylation, or otherwise interacting to form or break a bond, e.g., a covalent or non-covalent bond. A compound can include, for example, a fragment or analog of syndecan-4; a polypeptide other than syndecan-4, e.g., a randomly generated polypeptide which interacts with S4BP, or a small molecule. In a preferred embodiment, the method can include the steps of contacting the S4BP protein with the compound or agent under conditions which allow binding of the compound to the S4BP protein to form a complex and detecting the formation of a complex of the S4BP protein and the compound in which the ability of the compound to bind to the S4BP protein is indicated by the presence of the compound in the complex. Methods for identifying a compound or agent can be performed, for example, using a cell free assay. For example, S4BP can be immobilized to a suitable substrate, e.g., glutathione sepharose beads or glutathione derivatized microtitre plates, using a fusion protein which allows for S4BP to bind to the substrate, e.g., a glutathione-S-transferase/S4BP fusion protein.
In another embodiment, a compound or agent which interacts with an S4BP protein can be identified using a cell-based assay. These methods can include identifying a compound or agent based on its ability to modulate, e.g., inhibit or promote, a biological activity of S4BP. In a preferred embodiment, the compound modulates one or more of the following biological activities of S4BP: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 3) it interacts, directly or indirectly, with PKCxcex1; 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with paxillin; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
In another aspect, the invention features methods for identifying compounds which modulate S4BP nucleic acid expression. In a preferred embodiment, nucleic acid expression can be evaluated using a nucleic acid probe, e.g., a labeled probe, capable of hybridizing to an S4BP nucleic acid molecule, e.g., S4BP MRNA. In another preferred embodiment, S4BP nucleic acid expression, e.g., DNA expression, can be evaluated by contacting a compound with an S4BP nucleic acid molecule, e.g., a control region of a S4BP nucleic acid molecule, and evaluating S4BP transcription, in vitro or in vivo. S4BP transcription can be evaluated, for example, by detecting the production of S4BP protein, e.g., using an antibody, e.g., a labeled antibody, or by determining a cell activity, e.g., using a marker gene, e.g., a lacZ gene, fused to the control region of S4BP and following production of the marker.
The invention further features methods for identifying a compound or agent which modulates, e.g., stimulates or inhibits, the interaction of the S4BP protein with a target molecule, e.g., an adhesion receptor (e.g., syndecan-4) or a protein associated with cytoskeleton (e.g., actin, vinculin, paxillin), or a protein involved in a signaling pathway, e.g., a protein involved in adhesion dependent signaling events (e.g., focal adhesion kinase, serine-threonine kinase, GTP binding proteins such as PKCxcex1). In these methods, the S4BP protein is contacted, in the presence of the compound or agent, with the target molecule under conditions which allow binding of the target molecule to the S4BP protein to form a complex. An alteration, e.g., an increase or decrease, in complex formation between the S4BP protein and the target molecule as compared to the amount of complex formed in the absence of the compound or agent is indicative of the ability of the compound or agent to modulate the interaction of the S4BP protein with a target molecule.
A xe2x80x9cheterologous promoterxe2x80x9d, as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d or isolated xe2x80x9cpreparationxe2x80x9d of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 xcexcg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers to human and non-human animals. In preferred embodiments, the subject is a human e.g., a person having or diagnosed as at risk for having an S4BP related disorder. The term xe2x80x9cnon-human animalsxe2x80x9d of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, reptiles.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpure nucleic acidxe2x80x9d, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
xe2x80x9cSequence identity or homologyxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. To determine the percent homology of two amino acid sequences (e.g., SEQ ID NO:2) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., SEQ ID NO:2) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid xe2x80x9chomologyxe2x80x9d is equivalent to amino acid or nucleic acid xe2x80x9cidentityxe2x80x9d). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positionsxc3x97100). For example, if 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology or sequence identity.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to S4BP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to S4BP protein molecules of the invention. To obtain gapped alignments for comparison purposes. Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
A xe2x80x9cbiological activity of S4BPxe2x80x9d refers to one or more of the following activities: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 3) it interacts, directly or indirectly, with PKCxcex1; 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with paxillin; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; 9) it modulates cell spreading.
The term xe2x80x9csmall moleculexe2x80x9d, as used herein, includes peptides, peptidomimetics, or non-peptidic compounds, such as organic molecules, having a molecular weight less than 2000, preferably less than 1000.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more subject S4BP polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
xe2x80x9cUnrelated to an S4BP amino acid or nucleic acid sequencexe2x80x9d means having less than 30% sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occurring S4BP sequence disclosed herein.
A polypeptide has S4BP biological activity if it has one or more of the properties of S4BP disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the properties of S4BP disclosed herein.
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus. As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding an S4BP polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
As used herein, the term xe2x80x9chybridizes under stringent conditionsxe2x80x9d refers to conditions for hybridization and washing under which nucleotide sequences typically remain hybridized to each other. Preferably, the conditions are such that sequences which have at least about 60%, at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more sequence identity to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred example of stringent hybridization conditions are hybridization in 6xc3x97 sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 50-65xc2x0 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a xe2x80x9cnaturally-occurringxe2x80x9d nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural S4BP protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.